This project is focused on evaluating how the various DNA and protein immunogens affect the magnitude and breadth of anti-viral cell-mediated responses, and how these parameters contribute to protective immunity. The project will be done in collaboration with Project 3 using MHC typed macaques in trials and using macaque MHC class I tetramer reagents for assays as they become available. Specific Aim 1 will focus on comparing and optimizing assays of both CD8+ and CD4+ T cell responses. These studies will be conducted on macaques infected with SHIV 89.6, SHIV 89.6pd and live attenuated SIVmac239 delta nef to derive background data on the challenge stocks and for an infection and induces protective immunity. In Specific Aim 2, we will use selected assays to follow temporal anti-viral cell-mediated responses in vaccine trials. These analyses will be done both pre-and post-challenge and will compare the cell-mediated responses to those in infected animals. Comparative assays for CTL levels will be facilitated by using trial groups that include macaques with at least tone matched MHC class I allele. Specific Aim 3 will address the breadth and magnitude of the T cell responses raised by the different vaccine protocols in detail with an initial focus on gag epitopes. As MHC backgrounds become available, this analysis will be extended to pol and env for epitopes matching motifs identified for the MHC types in trials. The goals of this project are (i) to define whether broad and potent anti- viral cellular responses can be achieved and enhanced using DNA prime/protein boost protocols and (ii) to determine whether the magnitude and breadth of cell-mediated responses correlated with protective immunity.